(Support NIH/NCRR/P41 RR 01219 grant and NSF MCB 9420772 to B.F. McEwen) The strategy of the proposed research is to use same cell correlative video light microscopy, electron microscopic tomography (EMT) and 3-D immuno-electron microscopy to evaluate current hypotheses concerning the origin of polar ejection forces. These forces push objects away from the spindle poles and produce chromosome motion away from the pole. It is generally assumed that polar ejection forces arise from elongating spindle microtubules (Mts) pushing against objects they encounter. To test this hypothesis we are tracing Mts that impinge upon chromosome fragments that have been cut from chromosome arms by laser surgery. The prediction is that strongly ejecting fragments will have significantly more Mts than weakly or non-ejecting fragments. A second question is, do the Mts end at the fragment's surface, as predicted by a Mt pushing theory, or do they track along or through the fragmen t as pr edicted by a theory that ejection is generated by Mt motor molecules in the chromosome arms. Although primary cultures of newt lung cells are the tissue of choice for this project, newt lung tissue is seasonal and does not form confluent cultures with a significant mitotic index during the winter. For this reason, we attempted to perform the experiment on PtK1 cells. Mono-oriented chromosomes were created by destroying one of the sister kinetochores of a bio-oriented chromosome. After the mono-oriented chromosome migrated to one of the spindle poles, an acentric fragment was cut and tracked as it moved away from the spindle pole. Cells were fixed while the fragment was still moving and subsequently processed, embedded in Epon, and photographed on a CTEM. A serial-section reconstruction was made using Sterecon. A high-school student, Muniba Naqi, assisted in this project, and the resulting model of microtubules in association with the chromosome fragment was presented at Muniba' s school, along with a light-microscopy video of the ejecting chromosome. Ejected chromosome fragments were seen to interact laterally with several microtubules. This observation favors the hypothesis that Mt motors located in the chromosome arms generate the ejection force. However, it is difficult to trace Mts across successive serial thin sections. We then returned to working with mono-oriented chromosomes in newt lung cells because the spindle is large, giving more room for ejection, and there are fewer Mts, making it easier to tell which interactions are crucial. Despite problems with slowly-growing cell cultures, chromosomes were cut from three different anaphase-like prometaphase cells. One cell showed strong ejection and was fixed, while the other two did not show significant ejection. The fixed cell was stained for Mts with FITC and for chromosomes with Hoechst dye. We attempted to do a low-resolution 3-D reconstruction using optical deconvolution software, but this failed because the half-spindle had migrated underneath another cell, which contributed too much interfering staining. The cell was stained for Mts with a tertiary gold antibody (to the FITC secondary) and silver enhanced. It was then embedded and 0.65 micrometer-thick sections were cut. The silver staining was spectacular, but the section containing the ejected fragment was lost. Chromosomes from two other anaphase-like prometaphase cells were cut by laser microsurgery, but one showed no ejection and the other died during laser micro-surgery. Experience from this work was presented at the International Congress on EM at Cancun, Mexico in September. McEwen, B.F., Heagle, A.B., Cole, R., Matteyses, A., Rieder, C. L. (1998) Using correlative video light microscopy, laser microsurgery, and electron tomography to study polar ejection forces during mitosis in vertebrates. Electron Microscopy 1998 H. Benavides and M. Yacaman, Eds., Institute of Physics, Bristol and Philadelphia, Vol I, pp. 661-662.